The overall goal of the proposal is to understand the molecular mechanisms that ensure the production of functional gametes and healthy offspring. The proposal specifically focuses on meiotic silencing of unpaired chromatin in the nematode, Caenorhabditis elegans. Meiotic silencing is an epigenetic regulatory mechanism that has been described in mammals and nematodes as acting at the chromatin level to repress transcription. While the natural targets of meiotic silencing are the male sex chromosomes, the process will be activated to repress any unpaired chromosomes or chromosomal regions that arise due to mutation or chromosomal rearrangement during either female or male meiosis. Meiotic silencing is hypothesized to function in several ways to promote fertility and gamete quality. One hallmark of meiotic silencing in C. elegans, as in mammals, is the accumulation of a histone modification associated with transcriptional silencing, dimethylation of histone H3 on lysine 9 (H3K9me2). We have found meiotic H3K9me2 accumulation in C. elegans depends on the activity of several components of the small RNA machinery, including: EGO-1, an RNA-directed RNA polymerase (RdRP); CSR-1, a member of the Argonaute family of RNA-binding proteins; EKL-1, a Tudor domain protein; and DRH-3, a DEAD-box helicase. In addition to a role in meiotic silencing, our genetic evidence indicate that these four proteins participate in a functional pathway required for germline development and fertility. Our analysis of meiotic silencing in C. elegans provides a model for understanding the function of meiotic silencing in germline develoment, for tissue-specific formation of heterochromatin, and for chromatin regulation via small RNA-mediated mechanisms. Our data have allowed us to generate alternative models for the mechanism of H3K9me2 accumulation on unpaired chromatin. Here, we will test specific predictions of these models. In Aim 1, we will test alternative hypotheses for how known meiotic silencing components of the small RNA-mediated pathway (EGO-1, CSR-1, EKL-1, DRH-3) associate with paired or unpaired chromosomes. In Aim 2, we will test alternative predictions for how histone methyltransferase (HMTase) activity is recruited to unpaired chromosomes. These studies provide a complement to Aim 1. In Aim 3, we will determine the genomic sites of H3K9me2 accumulation on unpaired chromosomes and use this information to test for direct association of EGO-1, CSR-1, EKL-1, and/or DRH-3 with target loci. These studies will allow us to refine and extent the data obtained in Aim 1.